Project Summary/Abstract The overall objective is to define the molecular mechanisms through which Ca2+ homeostasis is perturbed in different brain diseases and how ion channels contribute to complex Ca2+-signaling intimately linked to neuronal damage and death. Our studies focus on inositol 1,4,5-trisphosphate receptor (IP3R) that is the primary Ca2+ release channel of the endoplasmic reticulum (ER), the cell's major Ca2+ storage organelle. Gating of IP3R is initiated by IP3 binding but tightly regulated by many additional signals, notably by interactions with pro- and anti-apoptotic Bcl-2 protein family members. Strong evidence suggests that de-regulation of IP3R inevitably leads to pathological conditions by ether an exaggerated cell death as in neurodegenerative diseases or escape from cell death as in some types of cancer. Despite recent progress in our understanding of how Bcl-2 proteins regulate the apoptotic switch, the structural basis for Ca2+ signaling disturbance in apoptosis via IP3R channels is largely unknown. There is a pressing need for atomic level structural information on how IP3R interacts with apoptotic proteins to coordinate its gating. To address this challenge, we extend our cryo-EM studies to recombinant full-length tetrameric IP3R1 purified from cultured cells (aim 1). The overarching goal of this aim is to push resolution limits in our cryo-EM studies of IP3R beyond 4.0 resolution recently achieved by our group for type 1 IP3R (IP3R1), purified from cerebellum. Using recombinant protein technology, we anticipate isolating more homogeneous channel sample than is likely present in native tissues. This will facilitate progress towards atomic-resolution structure determination and will allow to resolve protein regions that are missing in cryo-EM structure of native IP3R1 due to its genuine structural heterogeneity. Our goal is to solve structure of IP3R1 in complex with Bcl-2 protein to unveil the structural basis for the functional interactions of IP3R1 with modulatory apoptotic proteins, and to determine how the interplay between members of the Bcl-2 family sets the apoptotic threshold for cell life-or-death decision (aim 2). Structural studies will be complemented by functional experiments to establish physiological relevance of structural discoveries. The proposed studies are innovative since little is known at the atomic level about regulation of the IP3R channel by its binding partners, including apoptotic proteins. Because IP3R and Bcl-2 proteins are important to cell survival, adaptation and death processes, the proposed studies are significant to both our understanding of normal cellular physiology, and also to diseases associated with disturbed Ca2+ signaling and defective apoptosis, including neurodegeneration and cancer. Overall, this project represents the essential step towards atomistic descriptions of IP3R channel gating that will provide novel structural insights into critical IP3R channel functions and will serve as a platform for the development of future drugs targeting human IP3R.